This application is on prevention of African Trypanosomiasis, one of the most neglected diseases of Africa caused by parasitic African trypanosomes transmitted by tsetse. The absence of effective tools to curb infections in the mammal and the presence of animal reservoirs necessitate vector control to combat disease. We will investigate tsetse-trypanosome interactions that influence transmission dynamics. For transmission to occur, trypanosomes first establish infections in the midgut (MG) and then move to the fly's mouthparts to access and colonize the salivary glands (SG). The major barrier that eliminates parasites from the majority of flies occurs in the MG. We have shown that a parasite mediated manipulative process of vector's physiology transiently reduces midgut barrier integrity early in the infection to enable the parasites to bypass the peritrophic matrix (PM) barrier. At the core of this manipulative process is the mammalian parasite surface proteins, Variant Surface Glycoproteins (VSGs), shed into the gut lumen early in the infection process, which interfere with tsetse's PM synthesis acting through a microRNA (miR-275). Loss of PM integrity through a manipulative process again enables MG infecting parasites to re-enter into the lumen to colonize SG. We will use an interdisciplinary research plan to investigate: 1. The mechanisms that reduce PM efficacy and the different components of the parasite VSG protein that are responsible for this interference early in the infection process. We will also investigate the parasite components that enable PM reduction later in the infection process as parasites migrate from MG to SGs for transmission. We will perform vector and parasite transcriptomic profiling to discover potential mediators of the intra-organismal dialogue. 2. The role of the tsetse microRNA (miR275) in PM synthesis by identifying its downstream molecular targets through transcriptome and Riboseq profiling and by validating these targets using a dual-luciferase assay in a S2 cell line and through co-immunoprecipation assays. 3. Tsetse-parasite interactions in natural infections in the field to validate the parasite-vector dialogue we observe in the laboratory, and to determine the influence of PM modification on establishment of co- infections with multiple parasite species and strains. Using field flies, we will determine the course of parasite transmission processes to assess the epidemiological significance of PM barriers. Collectively, our studies will provide fundamental knowledge on adaptive and manipulative processes that influence vector competence and disease transmission in an important vector and will reveal potential targets for interference by transmission blocking strategies or paratransgenic applications to reduce disease.